Resistance to commonly used antibiotics has rendered many infections caused by Gram-negative bacteria very difficult to control, and sometimes the infections are untreatable. New antibiotics are urgently needed to control these infections; however, new antibiotics are becoming increasingly difficult to develop. The long-term goal of our program is to revive an old antibiotic class, the bicyclomycins, for several life-threatening pathogens. The present work focuses on a novel strategy for converting bicyclomycin from a largely bacteriostatic agent into a bactericidal one that will severely restric emergence of resistance. Previous work with Escherichia coli indicated that bicyclomycin, which targets the Rho transcription terminator, prevents removal of transcription elongation complexes from DNA when Rho-independent transcriptional terminators are absent. Replication forks then collide with the transcription complexes and generate DNA breaks. Although DNA breaks are potentially lethal, we recently found that treatment with bicyclomycin alone has little lethal effet on several Gram-negative bacteria. However, co-treatment of E. coli, Klebsiella pneumoniae, and Acinetobacter baumannii with bicyclomycin plus a second inhibitor of gene expression (e.g. bacteriostatic concentrations of chloramphenicol or tetracycline) either generates (E. coli) or dramatically increases (K. pneumoniae, A. baumannii) bicyclomycin-mediated lethality. We hypothesize that bicyclomycin treatment induces repair functions that severely limit bicyclomycin-mediated lethal activity; when inhibitors of translation are also present, bicyclomycin-induced repair is blocked, and bicyclomycin becomes lethal. We propose to identify and characterize genes involved in bicyclomycin-mediated cell death 1) by measuring the ability of bicyclomycin to kill E. coli mutants in the presence and absence of translation inhibitors and 2) by measuring the ability of bicyclomycin treatment to increase the abundance of proteins encoded by genes that affect bicyclomycin lethality. The primary outcome of the work will be a genetic understanding of the mechanism underlying bicyclomycin-mediated cell death. An important application will be identification of potential targets that can be used to fin small-molecule inhibitors for specifically generating/enhancing bicyclomycin-mediated lethality. Adding lethality to other favorable features of bicyclomycin is expected to revive interest in this distinct class of antimicrobial as a treatment for several serious Gram-negative infections.